Device and method for sampling of substances using alternating polarity

ABSTRACT

A method for sampling of a substance from a subject is provided, which comprises placing one or more sampling chambers on a collection site on a tissue surface on the subject; conducting electric current through the tissue to extract a substance from the subject in a first direction in one or more sampling chambers that functions alternatively as both an anode and cathode during the course of the method; reversing the polarity to cause direct current to flow in second direction opposite the first direction; and analyzing the sampling chamber or chambers for the concentration of a substance or a substance metabolite. There is also provided a device for sampling of a substance from an organism on continuously or intermittently using alternating polarity method based on the application of low intensity electric fields of alternating polarity across the skin (iontophoresis) to enhance the transport of a substance (such as glucose, lactic acid, pyruvic acid, and the like) from body tissues to a sampling chamber. The device comprises an electrical power supply; a transdermal system that contains one or more sampling chambers that function as both anode and cathode during the course of sampling; a means to alternates or reverses the polarity during the iontophoretic sampling; and means for analyzing for the concentration of a substance or a substance metabolite.

FIELD OF THE INVENTION

This invention relates to a device and method for sampling of substancesacross tissue using electrical energy of alternating polarity.

BACKGROUND OF THE INVENTION

Iontophoresis is employed for therapeutic topical treatment of humans orother organisms. Glikfeld et al., U.S. Pat. No. 5,279,543, describes theuse of iontophoresis to non-invasively sample a substance through skininto a receptor chamber on the skin surface. The Glikfeld deviceconsists of two chambers, each containing an electrically conductivemedium, which medium is in electrical contact with the skin. A positiveelectrode (anode) is disposed in one chamber, and a negative electrode(cathode) is disposed in the other. The two chambers are separated by anelectrically insulating material. In use, a constant or pulsed currentis generated by a power source between the electrodes through the skin.The direction of current flow never changes during sampling. Glikfelddoes not correlate the collected substance levels with blood substancelevels and does not address any problems associated with long termtransdermal monitoring of a substance.

The Lattin and Spevak U.S. Pat. No. 4,406,658, Tapper U.S. Pat. Nos.4,301,794, 4,340,047 and 5,224,927, Masaki U.S. Pat. No. 4,786,278,Masaki U.S. Pat. No. 4,792,702 and Masaki European Patent No. 230,153describe the iontophoretic delivery of a substance across tissue. Inaddition, these publications describe operation methods in which thepolarity of the applied electrical energy is reversed during delivery.None of these publications addresses extraction of substances fromtissue or any of the problems associated with such extraction.

The Tapper '794 and '047 patents address the irritation caused byiontophoretic delivery of ionized molecules through skin. In the Tapperapparatus, all delivery occurs at a single electrode. Tapper determinedthat the irritation can be eliminated if the polarity of the appliedcurrent is reversed for equal periods. A complete and equal polarityreversal eliminates the molecule delivery, however. Tapper compromisesby providing for a shorter period for the reverse direction and a longerperiod for the forward (substance delivery) direction.

The Tapper '927 patent discloses an apparatus that iontophoreticallydelivers a substance into the patient's skin from two source reservoirs,each attached to an electrode. The electrodes are connected to an ACpower source having a frequency in the range of 0.0027-10 Hz. Use of anAC power source minimizes the build-up of irritating chemicals atpositive and negative electrodes (e.g., HCl and H₂ O₂, respectively).Providing a source reservoir at both electrodes permits delivery fromone of the reservoirs in each phase of the AC signal, therebyeliminating the drawback of using a single source reservoir noted in theearlier Tapper patents.

Some alternating polarity methods for drug delivery, such as in theTapper '927 patent focus on prevention of pH changes. For many electrodesystems, water hydrolysis upon the application of current causes the pHto dramatically decrease at the anode and increase at the cathode. Thiscan cause skin irritation. Furthermore, the pH changes in the drug donorelectrodes can reduce the amount of ionized drug relative to non-ionizeddrug in the donor depending on the pKa of the drug. These patentspropose switching between the anode and cathode to prevent these pHchanges, reducing irritation and possibly increasing drug flux,especially of ionizable drugs.

The Lattin and Spevak '658 patent allows for delivery from bothelectrodes, thus doubling the potential delivery area. For drug deliveryapplications, more area means more deliverable drug per application of asingle device. However, for iontophoretic sampling applications, thereis no advantage to doubling the area because total delivery is not animportant factor. The amount that is sampled per area is more importantbecause it is the substance concentration that is to be measured, notthe total substance amount.

The Masaki patents disclose a device and a switch which can iontophoreseboth anions and cations by switching the polarity of the two electrodesduring the treatment cycle. In the Masaki '702 patent, the cations andanions come from a single source reservoir attached to one electrode.The other two patents do not disclose where the cation and anion sourcesare located. In any event, these patents teach nothing aboutiontophoretic sampling from skin into a reservoir.

Sibalis U.S. Pat. No. 5,013,293 describes an "electrophoretic" drugdelivery system in which a drug is delivered from a reservoir into thepatient's skin under the action of current applied to two electrodes,one of which is attached to the reservoir. The polarity of the currentmay be alternated periodically to control the amount of drug delivered.Sibalis does not disclose the use of iontophoresis for removing asubstance from a patient's tissue.

LaPrade U.S. Pat. No. 5,006,108 discloses alternating polarity for thedelivery of drugs in which the surface area of both electrodes (eachalternating between being the anode and cathode) is available to deliverthe drug. The LaPrade disclosure does not suggest that: (1) a singleelectrode may be used as a collector during iontophoretic extraction,with that electrode alternating between being an anode and a cathode;(2) two electrodes may act alternatively as anode and cathode duringiontophoretic extraction, or (3) iontophoresis can be used generally forremoving substances from tissue.

Some alternating polarity patents for drug delivery, such as the aboveLaPrade and Lattin patents, focus on increasing drug dosage with thesame size patch. The principle is that the available surface area fordelivery is larger for alternating polarity because each electrode canbe both an anode and a cathode, rather than one electrode being the"active" electrode and the other electrode taking up space, but nothelping drug delivery, as the "indifferent" electrode. This results inmore drug delivery, but would not aid in extraction where substanceflux, not "total" substance delivered, is the critical factor.

Teijin LTD, Japanese patent publication 4224770, discloses an apparatuswith electrodes for attachment to skin which has hydrous medium forelectrically contacting the electrodes, containing medically activeagent to be absorbed through the skin, and current polarity switchingmeans. Apparently, this patent is designed to reduce pH deviation.

Teijin LTD, Japanese patent publication 3045272, discloses a compactiontophoresis device which comprises electrodes containing medicine andapplied with high frequency AC for percutaneous administration and withthe objective of reducing skin irritation.

SUMMARY OF THE INVENTION

In the area of in vivo extraction of substances from tissue, correlationbetween measured concentrations of extracted substances must becorrelated with levels of the substance within the patient's tissueand/or blood stream. For example, studies have shown a need for avirtual continuous glucose monitor, which could potentially signal adiabetic of dangerously low or high blood sugar levels. The greater theflux of the extracted substance from the tissue into the substancecollector, the greater the accuracy with which the measured flux can becorrelated with the concentration of the substance within the tissueand/or blood stream. What is needed, therefore, is a way to decrease thedifference between the measured concentration of the extracted substanceand the concentration of the substance within the tissue and/or blood.

Standard iontophoretic electrodes generally involve either waterhydrolysis, which can cause undesirable pH changes, or a silver/silverchloride reaction in which silver chloride is converted to silver at thecathode, and silver is converted to silver chloride at the anode. Foreither of these systems, long term application of current can be aproblem due to the eventual depletion of the reactants. What is needed,therefore, is an iontophoretic sampling system and method that avoidsthis problem.

This invention is directed to a device and method for sampling of asubstance using electrical energy applied in alternating polarity.Alternating the polarity allows the reactions, such as silver/silverchloride reaction or the water hydrolysis reaction, to cycle back andforth, potentially lengthening the time that a particular electrodecould be used. In the non-invasive sampling method of the invention thepertinent parameter is the extraction of the desired substance per unitsurface area (flux), not the total extraction.

The invention includes a method for sampling of a substance from asubject, which comprises

(a) placing one or more sampling chambers on a collection site on atissue surface of a subject;

(b) conducting electrical current through the tissue in a first polarityto extract a substance from the subject into one or more samplingchambers;

(c) reversing the polarity of the electrical current during the courseof the method (i.e., changing an anode to a cathode and a cathode to ananode); and

(d) analyzing the sampling chamber or chambers for the concentration ofthe substance or a substance metabolite.

The invention also includes a sampling device comprising

(a) an electrical power supply means;

(b) a transdermal system that contains one or more sampling chambers forreceiving a sample that function as both anode and cathode during thecourse of iontophoretic sampling;

(c) means for reversing the polarity of the power source during theiontophoretic sampling; and

(d) means for analyzing the sampling chamber or chambers for theconcentration of the substance or a substance metabolite.

The invention also includes a substance monitor comprising:

first and second substance sampling chambers each containing substancecollection medium selected from the group consisting of water, salinesolutions, buffer solutions, and polyols;

a power supply means having a positive connector and a negativeconnector;

conductors and a switch electrically connecting the first and secondsampling chambers to the power supply means positive connector and thepower supply means negative connector, the switch having a firstposition in which the first sampling chamber is electrically connectedto the positive connector and the second sampling chamber iselectrically connected to the negative connector and a second positionin which the second sampling chamber is electrically connected to thepositive connector and the first sampling chamber is electricallyconnected to the negative connector.

The reversal of polarity during the sampling method of the invention hasadvantages over a prior standard iontophoresis method (non-alternatingdirect or intermittent current application) including: (1) there isunexpected enhancement of the correlation between blood andiontophoretically extracted samples under the alternating polaritymethod of the invention as compared to a non-alternating polarityprotocol, (2) there is a particularly unexpected normalized flux rateincrease over the use of direct current, and (3) the alternatingpolarity method of the invention enables continuous or intermittentapplication of current for Ag/AgCl or other electrodes without depletionof the AgCl plating or pH variations which occur for non-alternatingsystems.

The ionization state of the substance of the drug delivery materials ofthe prior art were dictated by the pH of the donor solutions. However,in the alternating polarity sampling method of the invention, theionization state of the extracted substance does not rely on the pH ofthe donor chamber; rather, the ionization state of the extracted speciesgoing from the body to the top of the skin is dictated by the pH of thebody, which is relatively constant.

The alternating polarity sampling method and device of the inventionpreferably uses low frequencies of about 0.1 Hz (one switch every 10seconds) to about 1 switch every hour. This is in contrast to the drugdelivery of the prior art, such as Tapper '794 and Masaki '351 whichdisclose that in a desire to reduce skin irritation, rapid, highfrequency pulsing, of either the same or alternating polarity, reducesskin irritation caused by iontophoresis.

These advantages are surprising considering the non-invasive nature ofthe method and device which produces these advantages when transportingsubstances that are beneath the skin to a collection reservoir on top ofthe skin while no part of the device penetrates into or beneath theskin.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1A is a block diagram of a substance monitoring device according tothe invention.

FIG. 1B shows an iontophoretic monitoring device useful for practicingthis invention in place on a patient.

FIG. 1C is an exploded drawing of the electrode/collection reservoirassembly illustrating the various layers thereof.

FIG. 1D is a cross-section of one of an electrode/reservoir assemblyFIG. 1C.

FIG. 2 is a graph of the results of a blood oral glucose toleranceglucose monitoring experiment on subject A, right arm, using a standardprotocol.

FIG. 3 is a graph of the results of a blood oral glucose toleranceglucose monitoring experiment on subject A, left arm, using analternating protocol.

FIG. 4 is a graph of the results of a blood oral glucose toleranceglucose monitoring experiment on subject B, right arm, using a standardprotocol.

FIG. 5 is a graph of the results of a blood oral glucose toleranceglucose monitoring experiment on subject B, left arm, using analternating protocol.

FIG. 6 is a graph of the results of a blood glucose monitoringexperiment using several cycles of direct current and a cycle ofalternating polarity.

FIG. 7 is the results of a blood glucose monitoring experiment with thesubject A of FIG. 3, under fasted conditions.

FIG. 8 is a graph of the results of a blood glucose monitoringexperiment with the subject A of FIG. 3, left arm, compensating fordrift at the cathode.

FIG. 9 is a graph of the results of a blood glucose monitoringexperiment with the subject A of FIG. 3 compensating for time lag anddrift at the cathode.

FIG. 10 is a graph of the results of a blood glucose monitoringexperiment with the subject A of FIG. 3 compensating for drift at theanode.

FIG. 11 is a graph of the results of a blood glucose monitoringexperiment with the subject A of FIG. 3 compensating for time lag anddrift at the anode.

FIG. 12 is a graph of the results of a blood glucose monitoringexperiment with the subject B of FIG. 5, left arm, compensating for timelag and drift at the cathode.

FIG. 13 is a graph of the results of a blood glucose monitoringexperiment with the subject B of FIG. 5, left arm, compensating for timelag and drift at the anode.

FIG. 14, subject D, right arm, demonstrates that the standard DCprotocol did not show this oscillation.

FIG. 15, subject D, left arm, showed sharp oscillation in theiontophoretically extracted glucose at the cathode as the physical siteof extraction was switched from one extraction site on the forearm tothe other during the alternating polarity protocol.

FIG. 16, subject B, left arm, shows that the oscillatory behavior issmoothed out by alternating the polarity every 7.5 minutes rather thanevery 15 minutes.

FIG. 17, subject B, right arm, shows the oscillatory behavior, similarto that observed in Example 4, for the 15-minute alternating polarityprotocol.

FIG. 18, subject A, right arm, demonstrates the dramatic enhancement influx at the cathode for a direct current protocol using pH 8.2 sodiumbicarbonate buffered saline solution compared to FIG. 2 for the sameSubject B using 0.45% sodium chloride solution.

FIG. 19, subject A, left arm, demonstrates the dramatic enhancement offlux at the cathode for the alternating polarity protocol using pH 8.2sodium bicarbonate buffered saline solution compared to FIG. 3 for thesame Subject B using 0.45% sodium chloride solution.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inart to which this invention is directed. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this invention, the preferred methods andmaterials are now described.

All patents and publications cited herein are incorporated herein byreference for the purpose of disclosing and describing information inconnection with which the patents and publications are cited.

By "conducting electrical current through the tissue to extract thesubstance into one or more sampling chambers/collection reservoirs" ismeant applying electrical energy of sufficient strength and duration tothe tissue surface of a subject in order to transport a substance or ametabolite from beneath the skin at a collection site on the surface ofthe tissue into a defined collection area. This includes the methodknown as iontophoresis. "Iontophoresis" as used herein means a method oftreatment to drive uncharged non-ionic materials and positive ornegative ions out of an organism through tissue. In conventionaliontophoresis two electrodes are placed in contact with the tissue. Oneor both of the electrodes is in a sampling chamber/collection reservoirto collect the substance extracted and a voltage is applied between thetwo electrodes. The sampling chamber/collection reservoir is provided atthe tissue surface to serve as a collector of material transported.

As used herein "sampling" or "monitoring" means the extraction of asubstance from an organism through the tissue. The tissue or skin can benatural or artificial tissue and can be of plant or animal nature, suchas natural or artificial skin, blood vessel tissue, intestinal tissue,mucosal epithelial tissue, and the like. The term "artificial" as usedherein means an aggregation of cells of monolayer thickness or greaterwhich are grown or cultured in vivo or in vitro and which function as atissue of an organism but are not actually derived, or excised, from apre-existing source or host. The subject/patient or host organism caninclude warm-blooded animals and is, particularly, a mammal, such asrats, mice, pigs, dogs, cats, cattle, sheep, horses, and especially ahuman being. When the tissue is human skin, the tissue surface is thestratum corneum surface, mucosal tissue as in the oral, nasal, orvaginal cavity or the like.

There is a need to sample and quantify or qualify bioactive-substances,such as glucose or a glucose metabolite, in a body, such as in theblood, for example, to monitor the presence of a endogenous biochemicalfor the purpose of diabetes diagnosis and treatment, forensicevaluation, or to follow, and preferably optimize the blood level of anadministered drug during a chemotherapeutic regimen. This preferablyneeds to be done without the invasive withdrawal of blood by a needleinto a collection reservoir/container. In some applications, e.g.diabetes treatment, for maximum benefit, sampling and quantificationalso should be performed substantially continuously and the measuredsubstance values need to correlate actual blood substance levels.

Substances that can be sampled from an organism include anything foundin the system of an animal, including a wide variety of body materialsintended for diagnosis, such as natural and therapeutically introducedmetabolites, hormones, amino acids, peptides and proteins, electrolytes,metal ions, suspected drugs of abuse, enzymes, tranquilizers,anesthetics, muscle relaxants, sedatives, antipsychotic agents,antidepressants, antianxiety agents, small drug molecules, and the like.Non-limiting representative examples of such materials include glucose,lactic acid, pyruvic acid, alcohols, fatty acids, glycols, thyroxine,estrogen, testosterone, theobromine, sucrose, galactose, uric acid,alpha amylase, choline, L-lysine, sodium, potassium, copper, iron,magnesium, calcium, zinc, potassium, citrate, morphine, morphinesulfate, heroin, insulin, steroids, neomycin, nitrofurazone,betamethasone, clonidine and clonidine HCl, acetic acid, alkaloids,acetaminophen, and amino acids. More than one substance can be sampledat one time. In one embodiment, the invention includes a continuousmonitoring of the levels of glucose or glucose metabolite from the body.

According to one embodiment, the invention is useful in continuousmonitoring of levels of a substance (glucose) or a substance (glucose)metabolite, e.g., lactic acid, from the body. The method can also beused for measurement of blood substance (glucose) levels in either asemi-continuous or a single measurement method. The method can bepracticed by a device that provides electrodes or other means forapplying electric current to the tissue at the collection site; one ormore collection reservoirs or sampling chambers to receive the substance(glucose); and a substance concentration measurement system. Acontinuous glucose monitoring method and apparatus according to thisembodiment is described in a patent application (Attorney Docket No.23000-20080.00) entitled "Continuous Transdermal Glucose MonitoringMethod and Apparatus" filed concurrently with this application andassigned to the assignee of this application, the disclosure of which isincorporated herein by reference. A preferred embodiment of the methodand apparatus are described in more detail below.

The method and device of the invention are useful in providingnon-invasive monitoring of the levels of a substance, such as glucose ora glucose metabolite, from the body. For example, the method and devicecan be used for the measurement of blood glucose levels in a diabeticsubject in either a semi-continuous or a single measurement method. Themethod can be practiced with a device of the invention based on theapplication of low intensity electric fields of alternating polarityacross the tissue/skin (iontophoresis) to enhance the transport ofmolecules, such as glucose, lactic acid, pyruvic acid, and the like,from body tissues to one or more collection reservoirs/samplingchambers.

According to the method of the invention, a collection reservoir isplaced at a collection site on a tissue surface of the patient, forexample, on the stratum corneum of the patient's skin or on the mucosalepithelium. Electrical energy is applied to the tissue at the collectionsite to move the substance from the tissue into the collectionreservoir. The collection reservoir is analyzed periodically to measurethe substance level concentration therein, and this measuredconcentration value is preferably correlated with the patient's bloodsubstance level. The steps are repeated to monitor the patient's bloodsubstance level, preferably substantially continuously, and to trackchanges in the patient's blood substance level.

In a preferred embodiment of the method, two electrodes provide theelectrical energy to the tissue at the collection site or sites. Twocollection reservoirs are provided, one at or near each electrode. Theapplied electrical current is preferably in the range of about 0.01 toabout 2 mA/cm². In applying the electrical energy, the polarity of theelectrodes is alternated, e.g., at a rate in the range of about 0.1 Hz(one switch every 10 seconds) to about 1 switch every hour, so that eachelectrode is alternately a cathode or an anode. The substance inquestion (e.g., glucose) is collected in both reservoirs, but is mostpreferably measured in the reservoir acting as the cathode. In otherwords, the reservoir in which glucose concentration is monitoredalternates as the polarity of the electrodes alternates. The actualfrequency of the polarity switch may need to be optimized for thesubstance being extracted and measured.

The reversal of polarity during sampling has advantages, including: (1)an unexpected enhancement of the correlation between blood and/or tissueconcentrations and iontophoretically extracted samples under thealternating polarity method of the invention as compared to anon-alternating polarity protocol, (2) a particularly unexpectedincrease in the normalized flux rate, and (3) the avoidance of depletionof the AgCl plating on AgCl electrodes or pH variations which occur fornon-alternating systems.

The polarity switching can be manual or automatic. The polarityswitching can be of any frequency, especially at frequencies betweenabout 1 cycle per 20 seconds to about 1 cycle per 4 hours, preferablybetween about 1 cycle per 20 seconds to about 1 cycle per 2 hours, ormore preferably between about 1 cycle per minute to about 1 cycle per 2hours, or between about 1 cycle per 10 minutes to about 1 cycle perhour, and especially is about 1 cycle per half hour. By cycle is meantone interval of current in each direction. The application of electricalenergy in a given cycle may cease during analysis of the collectionmedium.

In the preferred method, part or all of the collection medium isextracted from the collection reservoir and analyzed for concentrationof the substance in question. One preferred analysis method is highpressure liquid chromatography (HPLC), although other analysis methodsmay be used, such as pulsed amperametric detection.

To provide useful information regarding concentration levels of thesubstance in the patient's blood and/or tissue, the substanceconcentrations detected in the collection medium must be correlated withthe tissue and/or blood levels. Concentration in the collection mediumis computed in flux terms, such as nmoles/cm² ·hr. This information mustbe translated into corresponding values (expressed, e.g., in terms ofmg/dl for blood substance concentrations).

For example, correlation between blood glucose concentration and measureglucose flux may be expressed in terms of four variables: the time lagbetween blood glucose concentration values and measured glucose fluxvalues; linear drift over time of the measured glucose flux values; andthe slope and intercept (i.e., the low-end blood glucose value belowwhich no glucose flux is detected) of a line relating time lag anddrift-corrected glucose flux data to blood glucose concentration.Correlation between measured glucose flux and actual blood glucoseconcentration is accomplished through a calibration procedure whichrelates blood glucose data and glucose flux data.

Time lag is defined as the constant time shift between blood glucose andglucose flux curves that achieves a best fit in curve shape along thetime axis within predefined tolerances. The amount of time shift may bedetermined manually or automatically with the aid of a computer or otherprocessor using known curve shape comparison algorithms. Time shift isillustrated in FIGS. 8 & 9 and FIGS. 10 & 11.

Drift is defined as the linear change with time of the measured glucoseflux for a given blood glucose concentration. Drift is computed byshifting the plotted glucose flux curve with respect to the bloodglucose concentration curve along the glucose flux axis by a constantmultiple of the time variable to achieve a best fit. The value of theconstant multiple may be computed in many different ways. For example,experimental data taken from a given subject at given times under"baseline" conditions (i.e., the patient is fasting and the bloodglucose level is therefore constant) may be subtracted from measuredglucose flux data point by point during the oral glucose tolerance testat the same times for the same patient.

As another example, a linear fit (obtained, e.g., from a least squareslinear regression) of the baseline data for a given patient may besubtracted from experimental data obtained during the oral glucosetolerance test. Measured glucose flux values can also be correctedwithout first obtaining baseline values. One approach is to estimate thevalue of drift time constant, adjust for drift based on the estimate,and determine the correlation between drift-corrected measured valuesand measured blood glucose values. The estimated drift time constant canthen be increased or decreased, and the correlation recalculated, untilan acceptable tolerance between measure glucose flux values and bloodglucose values is achieved.

The drift time constant may also be computed by measuring a differentphysiological parameter, such as the electrical resistance of thepatient's skin (which may change with time in the same manner as theglucose flux value drift) or a parameter that remains relativelyconstant over time, such as the pH of the patient's blood.

Finally, the slope and intercept of the relationship between the timeand drift corrected values is computed using a linear best fit of thedata. These steps may be performed by hand or by a suitably programmedprocessor or computer.

Other correlation variables and correlating relationships may bedetermined for other sample substances.

The sampled substance may be present in nonsystemic amounts in thestratum corneum prior to application of the monitoring method anddevice. A time delay may therefore be provided between the start of thesampling and the time of analysis.

The method of this invention may be performed substantially continuouslyfor an indefinite period of time. The method of the invention may alsoinclude the step of treating the patient in response to the determined(i.e., correlated) systemic concentration of the substance in thepatient.

The collection reservoir/sampling chamber of the device of the inventioncan be in the form of (1) a single chamber with an electrode, whichalternates polarity, with an indifferent counterelectrode which alsoalternates polarity, with the electrode in the sampling chamber actingas anode, cathode or both during the course of sampling, (2) twochambers in which the anode and cathode chambers are on alternate sitesas the polarity alternates or (3) two or more chambers in which thepolarity alternates during the treatment allowing for signal averagingbetween cathode and anode.

The collection reservoir/sampling chamber for receiving the sample ofthe target substance from the subject can be in the form of adhesivepatch enclosing the electrode means. A wide variety of adhesives forpatches are known in the iontophoresis art.

FIG. 1A shows in block diagram form a monitor according to thisinvention. The monitor includes a collection reservoir 100 containing acollection medium. A pair of electrodes 102 and 104 connected to a powersupply 106 having positive and negative connectors 107 and 108,respectively, via conductors 109 and 110 and a switch 112 providecurrent for transport of the sampled substance from a tissue surfaceinto reservoir 100. The switch 112 may be controlled manually or by anautomatic controller 113. An analyzer 114 associated with the monitormeasures the substance concentration in collection reservoir 100 andcomputes a substance flux. A calibrator 116 correlates the computedsubstance flux with the patient's systemic concentration.

FIGS. 1B-1D are drawings of a preferred device for practicing the methodof this invention. As shown in FIG. 1B, two integral electrode/reservoirassemblies 10 are attached to collection sites 12 on a tissue surface 14of a patient. The electrode/reservoir assemblies 10 are connected to apower supply 20.

FIG. 1C is an exploded view of the electrode/reservoir assembly 10.Element 1 is a release liner which is a protective layer for theadhesive of the skin contacting surface of the patch; element 2 is asample port which can be a tube through which samples are removed;element 3 is the top layer, which can be a polymeric layer, which formsthe upper housing for the collection reservoir/sample chamber and makesit possible to view the condition of the electrode as the treatmentprogresses; element 4 is a foam layer coated on one side with anadhesive top layer, which prevents contact of the electrode with theskin and serves as the walls of the collection reservoir/sample chamber;element 5 or 6 is an electrode which serves as either the cathode (i.e.,electron donor) or the anode (i.e., electron receiver) depending on thepolarity of the electrodes; element 7 is a transfer adhesive layer whichholds the electrode 5 and the sample port 2 in place; and element 8 is aprotective layer positioned between the electrode 5 and the skin toprevent the electrode from contacting the skin.

As illustrated, layers 4 and 7 each comprise part of the housing of thecollection reservoir and allow for the collection reservoir to be opento the skin.

The release liner 1 is a protective layer for the adhesive of the skincontacting surface of the patch. This liner can be any suitable flexiblematerial conventional known in the art. It should be strong enough notto be torn away by normal movement but easily removed by the applicationof some manual effort.

The sample port 2 can be a tube through which samples are removed.

The top layer 3 can be any flexible material conventionally known in theart, such as a polymeric layer, which forms the upper wall of thehousing of the collection reservoir/sample chamber and is preferablytransparent, which makes it possible to view the condition of theelectrode as the treatment progresses. Any transparent polymericmaterial conventionally used as the top layer in transdermal patches canbe used. Preferably, the top layer is made of polyester or preferablypolyethylene.

The foam layer 4 is any medical grade foam coated on one side with anadhesive (foam tape) which prevents contact of the electrode with theskin and serves as the walls of the sample chamber. Any foam tape whichis conventionally used in transdermal patches which is compatible withthe tissue and material to be sampled can be used, preferably the foamtape is a closed-cell polyolefin foam coated on one side with anacrylate adhesive, such as 3M #9773.

The electrode 5 can be made of Ag/AgCl or any other electrode material.It is attached to the power source via electrical wires/leads.

The transfer adhesive layer 7 which is a transfer adhesive layer whichholds the electrode and the sample port in place. The adhesive can beany adhesive conventionally known in the art which can hold the layerstogether with the electrodes between. For example, the transfer adhesivecan be 3M Product #1524, medical transfer tape.

The protective layer 8 is positioned between the electrode 5 and theskin to prevent the electrode from contacting the skin. The protectivelayer is a polyurethane film coated on one side with an adhesive layerwhich keeps it in contact with the electrodes. Any conventional adhesivewhich is compatible with the skin can be used. For example, theprotective layer is an Acutek tape (Flexcon-Deraflex NRU 100 clear H566spec 50K-9 (nylon reinforced cast polyurethane film coated with anacrylic adhesive).

FIG. 1D is a cross-section of one of an electrode/reservoir assembly 10in place at the collection site 12. A collection reservoir/samplingchamber 16 is preferably filled with an electrically conductivemedium/fluid. Any fluid which is capable of conducting electric currentbetween the electrodes can be used. Suitable fluids include water,aqueous saline solutions and preferably aqueous buffered salinesolutions having a pH in the range of from about 4 to about 9. Apreferred fluid is an aqueous sodium bicarbonate buffered sodiumchloride solution having a pH of from about pH 7 to about pH 9.Obviously, the fluid is one that is inert to the material desired to besampled (i.e., glucose) and compatible with skin. The collection mediumprovides an electrical circuit connection between electrode 5 and thetissue surface.

Instead of the collection reservoir described above, a collection matrixmay be provided, as in known in the art. Also, electrodes in directcontact with the skin may be used in place of the electrodes immersed inconductive fluid as described above. The electrodes may be placed at thecollection site or adjacent to the collection site.

Preferably, power supply 20 comprises a means for reversing the polarityof the power source during the sampling, such as a manual or automaticprogrammable switch. One suitable power supply is the Iomed Phoresor2iontophoretic power supply.

One of skill in the art can recognize that there is variation betweensubjects or even for a given subject and for use of differentiontophoretic device designs, surface area, current density, and thelike as to background (non-systemic) substance concentrations, time lagand drift. These can be determined by standard methods and thecorrelation of the data adjusted for background substanceconcentrations, time lag and drift for an individual and/or device.

In one embodiment the invention includes a substance monitor comprising:

first and second substance sampling chambers each containing substancecollection medium selected from the group consisting of water, salinesolutions, buffer solutions, and polyols;

a power supply means having a positive connector and a negativeconnector;

conductors and a switch electrically connecting the first and secondsampling chambers to the power supply means' positive connector and thepower supply means' negative connector, the switch having a firstposition in which the first sampling chamber is electrically connectedto the positive connector and the second sampling chamber iselectrically connected to the negative connector and a second positionin which the second sampling chamber is electrically connected to thepositive connector and the first sampling chamber is electricallyconnected to the negative connector.

Preferably, the monitor is one in which the first and second samplingchambers each further comprises an electrode in electrical communicationwith the collection medium and with the power supply through a conductorand the switch.

The method of the invention is preferably one wherein the tissue surfaceis a stratum corneum surface of the patient's skin, the analysis stepcomprising the step of delaying the time between the applying step andthe analyzing step to allow for depletion of non-systemic substance(glucose) in the stratum corneum.

The delaying step can comprise delaying performance of the analyzingstep for about one hour after commencing the applying step.

The method of the invention preferably is one in which the collectionreservoir contains a collection medium, the analyzing step comprisingthe step of extracting at least a portion of the collection medium fromthe collection reservoir.

The method of the invention can further comprise the step of providingcorrelation data between collection reservoir substance (glucose) levelsand patient's blood substance (glucose) levels.

The method of the invention can also include the correlating stepcomprising calculating substance (glucose) flux from the collection siteinto the collection reservoir.

The method of the invention preferably comprises the method wherein thesteps of placing the collection reservoir on a tissue surface, applyingthe electrical energy to the tissue, analyzing the collection reservoirfor collected substance concentration, and correlating the analyzedsubstance concentration with systemic level are performed substantiallycontinuously for about 2 hours to about 72 hours, preferably from about16 hours to about 24 hours.

EXAMPLES

The following examples are provided to illustrate the invention butshould not be regarded as limiting it in any way.

Example 1

Blood glucose monitoring experiments were performed in which acomparison was made between a standard iontophoretic protocol consistingof direct current (DC) between an anode site and a cathode site versusan iontophoretic protocol of this invention in which the electrodes actas both the anode and the cathode (alternating polarity, AP) during thecourse of extraction. Two iontophoretic extractions were performedsimultaneously on human subjects, one using the standard (DC) protocolon the right arm and the other using the protocol of this invention (AP)on the left arm.

In these experiments, two adhesive sampling patches, each with Ag orAgCl electrodes inserted into the aqueous sampling chamber of thepatches, were applied to each forearm. The iontophoretic patches had acollection surface area of 2.85 cm² per patch and a collection reservoirvolume 0.4 mL per patch. The chambers were filled with 0.45% sodiumchloride in water, U.S.P.

For the direct current or DC protocol (the standard protocol), a currentof 0.32 mA/cm² was applied for about 15-minutes, followed by a 5-minuteinterval in which the saline was removed for analysis and then replacedwith fresh saline. During the 5-minute rest period, blood glucose wasmeasured by a standard finger prick (One Touch Basic, LifeScan,Milpitas, Calif.) method for comparison to extracted glucose. The15-minute iontophoresis and 5-minute rest/sampling procedure wasrepeated over a period of 3 to 5 hours. The extract samples wereanalyzed by HPLC.

The method of this invention (alternating polarity, AP) was identical tothe standard protocol, except that the polarity of the power supply wasswitched during each 5-minute rest period. In this way, the cathodechamber of one 5-minute period became the anode chamber of the next5-minute period, and vise versa.

During the method, an oral glucose tolerance test was conducted on thenon-diabetic subjects who had fasted prior to the experiment for 12hours overnight. One hour into the test, 75 g of glucose wasadministered orally. Blood glucose levels typically increased from 80 to150 mg/dL. The results of the test are set forth in FIGS. 2-5.

These figures are graphs comparing the results of the standard protocoland the alternating polarity method of the invention performedsimultaneously on the same subject. FIGS. 2 and 4 show the standard (DC)method, which was performed on the right arms of subjects A and B,respectively, and FIGS. 3 and 5 show the alternating polarity method ofthe invention, which was performed on the left arms of subjects A and B,respectively. In each figure, blood glucose concentration in mg/dL isgiven versus time in minutes. The "x" is for blood glucose, solidtriangle is for iontophoretic glucose at the anode, and the solid squareis for iontophoretic glucose at the cathode.

As can be seen by comparing the FIGS. 2 and 4 with FIGS. 3 and 5,respectively, the alternating polarity method of the invention gavebetter correlation, particularly for the anode response.

The above results demonstrate the improvement in sampling profiles fromalternating polarity, or the use one or more sampling electrodes thatact, alternatively, as anode and cathode during a cycling period duringthe course of the extraction, as compared to the direct current (DC)standard mode of iontophoresis.

Example 2

A continuous iontophoretic extraction was conducted on subject C.Iontophoresis was administered and glucose was monitored as described inExample 1 above, with the following modification. The 15-minuteextraction, 5-minute rest period sampling procedure was applied DC for 8sampling periods. On the 9th sample period, the current was switched toan alternating polarity protocol of a switch every 15-minutes. Theresults are set forth in FIG. 6. There was a sharp and dramatic increasein flux as the protocol was switched from direct current to alternatingpolarity.

Example 3

The following are results of tests in which the data from thealternating polarity protocol is corrected for time lag and/or drift asdiscussed below.

A continuous iontophoretic extraction was conducted on subject A as inFIG. 2, but in a protocol in which the subject had fasted 12 hoursovernight prior to the start of the experiment and during the experiment(no oral glucose tolerance test). Iontophoresis was administered usingthe alternating polarity method of this invention and glucose wasmonitored as described in Example 1 above. The results are shown in FIG.7.

FIG. 7 demonstrates that there was a drift in the anode and cathodefluxes for subject A using alternating polarity.

FIG. 8 is a graph of the results of a blood glucose monitoring as shownin FIG. 3 of subject A, but with the flux values for the cathode in FIG.7 subtracted from the flux values for the cathode in FIG. 3.

FIG. 9 is a graph of the results of a blood glucose monitoring as shownin FIG. 3 of subject A, but with the cathode flux adjusted for a 20minute time lag and the drift as in FIG. 8.

FIG. 10 is a graph of the results of a blood glucose monitoring as shownin FIG. 3 of subject A, but with the flux values for the anode in FIG. 7subtracted from the flux values for the anode in FIG. 3.

FIG. 11 is a graph of the results of a blood glucose monitoring as shownin FIG. 3 of subject A, but with the anode flux adjusted for a 40 minutetime lag and the drift as in FIG. 10.

FIG. 12 is a graph of the results of a blood glucose monitoring as shownin FIG. 5 of subject B, but with the cathode flux adjusted for a 20minute time lag and an empirically fitted linear drift.

FIG. 13 is a graph of the results of a blood glucose monitoring as shownin FIG. 5 of subject B, but with the anode flux adjusted for a 40 minutetime lag and an empirically fitted linear drift.

Example 4

Two iontophoretic extractions were performed simultaneously on subjectD, one using the standard (DC) protocol on the right arm and the otherusing the protocol of this invention (AP) on the left arm. Iontophoresiswas administered and glucose was monitored as described in Example 1above. The results of the test are set forth in FIGS. 14 and 15.

In FIG. 15, Subject D showed sharp oscillation in the iontophoreticallyextracted glucose at the cathode as the physical site of extraction wasswitched from one extraction site on the forearm to the other during thealternating polarity protocol.

FIG. 14 demonstrates that the standard DC protocol did not show thisoscillation.

In total, 12 normal subjects were tested as in Examples 1 and 4. Theresults are summarized in Table 1 below.

                  TABLE 1                                                         ______________________________________                                                  Direct                                                                              Current  Alternating                                                                             Polarity                                             Cathode                                                                             Anode    Cathode   Anode                                      ______________________________________                                        Good tracking                                                                             0        5*      7       4                                        Oscillation 0       0        5       6                                        Long Lag (>60 min)                                                                        5       1        0       2                                        No response 7       6        0       0                                        TOTAL       12      12       12      12                                       ______________________________________                                         Good = tracking with drift = r.sup.2 ≧ 0.5, lag ≦ 40 min        Long lag = r.sup.2 ≧ 0.5, lag ≧ 60 min                          Oscillation = qualitative tracking with oscillation (r.sup.2 ≧ 0.5     lag ≦ 40 min)                                                          No response = r.sup.2 ≦ 0.5                                            *Anode tracking, but low magnitude of flux                               

Example 5

A continuous iontophoretic extraction was conducted on one arm ofsubject B, as in FIGS. 4 and 5, except with a polarity switch every 7.5minutes instead of every 15 minutes. Iontophoresis was administered andglucose was monitored as described in Example 1 above, with thefollowing modification. Halfway through the 15-minute extraction period,the polarity was switched while the saline solution remained in thesample chambers. The sample was removed at the end of the 15-minutecycle. In this way, each electrode acts as the cathode for 7.5 minutesand the anode for 7.5 minutes. This method was performed simultaneouslyon the other arm with the 15-minute alternating polarity protocol asdescribed in Example 1 to the same subject B. The results are set forthin FIGS. 16 and 17.

FIG. 17 shows the oscillatory behavior, similar to that observed inExample 4, for the 15-minute alternating polarity protocol.

FIG. 16 shows that the oscillatory behavior is smoothed out byalternating the polarity every 7.5 minutes rather than every 15 minutes.The 7.5-minute protocol administered to each site an equal period ofanode and cathode extraction. The correlation of extracted and bloodglucose was thereby improved. This demonstrates that one or moreelectrodes can act as anode, cathode, or both during the course of theexperiment.

Example 6

Two iontophoretic extractions using a modified buffer solution wereperformed simultaneously on same subject A as in FIG. 2 and 3, oneextraction using the standard (DC) protocol on the right arm and theother using the protocol of this invention (AP) on the left arm.Iontophoresis was administered and glucose was monitored as described inExample 1 above, with the following modification. Sodium bicarbonatebuffer solution, 5% U.S.P, pH 7-9, mixed in a 1:9 ratio with 0.45%sodium chloride U.S.P. was used as the receiver solution instead of0.45% sodium chloride. The results are set forth in FIGS. 18 and 19.

FIG. 18 demonstrates the dramatic enhancement in flux at the cathode fora direct current protocol using pH 8.2 sodium bicarbonate bufferedsaline solution compared to FIG. 2 for the same Subject B using 0.45%sodium chloride solution.

FIG. 19 demonstrates the dramatic enhancement of flux at the cathode forthe alternating polarity protocol using pH 8.2 sodium bicarbonatebuffered saline solution compared to FIG. 3 for the same Subject B using0.45% sodium chloride solution. Further, a comparison of FIGS. 18 and 19indicates that there was enhanced anode extraction for the sodiumbicarbonate solution in the alternating polarity method compared to thedirect current method.

What is claimed is:
 1. A method for sampling of a substance from asubject which comprises:(a) placing a device comprising a first andsecond sampling chamber each sampling chamber comprising an electrode inelectrical communication with the tissue on a collection site on atissue surface on the subject; (b) conducting electrical current throughthe tissue in a first polarity to the collection site to extract asubstance from the subject into the first sampling chamber; (c)analyzing the first sampling chamber for the concentration of thesubstance or a substance metabolite; (d) reversing the polarity of theelectrodes in the first and second sampling chamber and conductingelectrical current through the tissue in a second opposite polarity tothe collection site to extract a substance from the subject into thesecond sampling chamber; (e) analyzing the second sampling chamber forthe concentration of the substance or a substance metabolite.
 2. Themethod of claim 1 wherein the polarity reversing step comprises the stepof using a switch to reverse polarity.
 3. The method of claim 1 whereinthe step of reversing polarity is performed at a frequency of from about1 cycle per 20 seconds to about 1 cycle per 4 hours.
 4. The method ofclaim 3 wherein the frequency of polarity switching is from about 1cycle per 20 seconds to about 1 cycle per 2 hours.
 5. The method ofclaim 4 wherein the frequency of polarity switching is from about 1cycle per minute to about 1 cycle per 2 hours.
 6. The method of claim 5wherein the frequency of polarity switching is from about 1 cycle per 10minutes to about 1 cycle per hour.
 7. The method of claim 6 wherein thefrequency of polarity switching is about 1 cycle per half hour.
 8. Themethod of claim 1 wherein the sampling chamber comprises an Ag/AgClelectrode, the conducting step comprising conducting current through theelectrode.
 9. The method of claim 1 wherein the alternating polaritycurrent is applied continuously.
 10. The method of claim 1 wherein thealternating polarity current is applied intermittently.
 11. The methodof claim 1 wherein the step of placing a sampling chamber on acollection site on a tissue surface comprises the step of placing asampling chamber on a collection site on a stratum corneum surface. 12.The method of claim 1 wherein the step of placing a sampling chamber ona collection site comprises the step of placing a sampling chambercomprising an adhesive patch enclosing electrodes and open to the tissuesurface.
 13. The method of claim 1 further comprising the step ofplacing electrodes on a tissue surface before the step of conductingelectrical current.
 14. The method of claim 13 wherein the electrodesare placed at the collection site.
 15. The method of claim 13 whereinthe electrodes are placed adjacent to the collection site.
 16. Themethod of claim 13 wherein the step of placing electrodes comprises thestep of placing on the tissue surface electrodes which are integral withthe sampling chamber.
 17. The method of claim 1 wherein the samplingchamber contains a collection medium, the step of conducting electricalcurrent to the collection site comprising the step of applyingelectrical energy to the collection medium.
 18. The method of claim 1wherein the step of conducting electrical current comprisesiontophoresis.
 19. The method of claim 1 wherein the step of conductingelectrical current comprises applying a current density in the range of0.01 to 2 mA/cm².
 20. The method of claim 1 wherein the step ofconducting electrical current comprises the step of ceasing theapplication of electrical energy during the analyzing step.
 21. Themethod of claim 1 wherein the analyzing step comprises the step of usingpulsed amperometric detection.
 22. The method of claim 1 wherein theanalyzing step comprises the step of using high performance liquidchromatography (HPLC).
 23. The method of claim 1 wherein the samplingchamber contains a collection medium, the analyzing step comprising thestep of extracting at least a portion of the collection medium from thesampling chamber.
 24. The method of claim 1 further comprisingcorrelating the concentration determined in step (c) with a bloodsubstance level.
 25. The method of claim 24 further comprising the stepof providing correlation data between a sampling chamber substance leveland the blood substance levels.
 26. The method of claim 25 wherein thecorrelating step comprises calculating substance flux from thecollection site into the sampling chamber.
 27. The method of claim 26wherein the correlating step also comprises compensating for time lagbetween blood substance level values and substance flux.
 28. The methodof claim 27 wherein the correlating step also comprises compensating forsubstance flux value drift.
 29. The method of claim 1 wherein the stepsare performed substantially continuously for about 2 hours to about 72hours.
 30. The method of claim 1 further comprising the step of treatingthe patient in response to the substance level determined. 10 minutes toabout 1 cycle per hour.
 31. An iontophoresis sampling devicecomprising(a) an electrical power supply; (b) a transdermal system thatcontains a sampling chamber for receiving a sample, wherein the samplingchamber is connected with said electrical power supply; (c) means forreversing polarity of the sampling chamber, wherein the sampling chamberfunctions as both an anode and a cathode during iontophoretic sampling;and (d) means for analyzing a concentration of a substance or asubstance metabolite received in the sampling chamber.
 32. The device ofclaim 31 wherein the means for reversing comprises means for reversingthe polarity of the sampling chamber at a frequency of from about 1cycle per 20 seconds to about 1 cycle per 4 hours.
 33. The device ofclaim 32 wherein the frequency of polarity switching is from about 1cycle per 20 seconds to about 1 cycle per 2 hours.
 34. The device ofclaim 33 wherein the frequency of polarity switching is from about 1cycle per minute to about 1 cycle per 2 hours.
 35. The device of claim34 wherein the frequency of polarity switching is from about 1 cycle per10 minutes to about 1 cycle per hour.
 36. The device of claim 35 whereinthe frequency of polarity switching is about 1 cycle per half hour. 37.The device of claim 31 wherein the sampling chamber comprises an Ag/AgClelectrode.